A role for adenine nucleotides in the sensing mechanism to purine starvation in Leishmania donovani

Purine salvage by Leishmania is an obligatory nutritional process that impacts both cell viability and growth. Previously, we have demonstrated that the removal of purines in culture provokes significant metabolic changes that enable Leishmania to survive prolonged periods of purine starvation. In order to understand how Leishmania sense and respond to changes in their purine environment, we have exploited several purine pathway mutants, some in which adenine and guanine nucleotide metabolism is uncoupled. While wild type parasites grow in any one of a variety of naturally occurring purines, the proliferation of these purine pathway mutants requires specific types or combinations of exogenous purines. By culturing purine pathway mutants in high levels of extracellular purines that are either permissive or non‐permissive for growth and monitoring for previously defined markers of the adaptive response to purine starvation, we determined that adaptation arises from a surveillance of intracellular purine nucleotide pools rather than from a direct sensing of the extracellular purine content of the environment. Specifically, our data suggest that perturbation of intracellular adenine‐containing nucleotide pools provides a crucial signal for inducing the metabolic changes necessary for the long‐term survival of Leishmania in a purine‐scarce environment.     

Population structure of a vector‐borne plant parasite

Parasites are among the most diverse groups of life on Earth, yet complex natural histories often preclude studies of their speciation processes. The biology of parasitic plants facilitates in situ collection of data on both genetic structure and the mechanisms responsible for that structure. Here, we studied the role of mating, dispersal and establishment in host race formation of a parasitic plant. We investigated the population genetics of a vector‐borne desert mistletoe (Phoradendron californicum) across two legume host tree species (Senegalia greggii and Prosopis velutina) in the Sonoran desert using microsatellites. Consistent with host race formation, we found strong host‐associated genetic structure in sympatry, little genetic variation due to geographic site and weak isolation by distance. We hypothesize that genetic differentiation results from differences in the timing of mistletoe flowering by host species, as we found initial flowering date of individual mistletoes correlated with genetic ancestry. Hybrids with intermediate ancestry were detected genetically. Individuals likely resulting from recent, successful establishment events following dispersal between the host species were detected at frequencies similar to hybrids between host races. Therefore, barriers to gene flow between the host races may have been stronger at mating than at dispersal. We also found higher inbreeding and within‐host individual relatedness values for mistletoes on the more rare and isolated host species (S. greggii). Our study spanned spatial scales to address how interactions with both vectors and hosts influence parasitic plant structure with implications for parasite virulence evolution and speciation.     

Beak of the pinch: anti-parasite traits are similar among Darwin’s finch species

Darwin’s finches are an iconic example of adaptive radiation. The size and shape of the beaks of different finch species are diversified for feeding on different size seeds and other food resources. However, beaks also serve other functions, such as preening for the control of ectoparasites. In diverse groups of birds, the effectiveness of preening is governed by the length of the overhanging tip of the upper mandible of the beak. This overhang functions as a template against which the tip of the lower mandible generates a pinching force sufficient to damage or kill ectoparasites. Here we compare feeding versus preening components of the beak morphology of small, medium, and large ground finches that share a single parasite community. Despite adaptive divergence in beak morphology related to feeding, the three species have nearly identical relative mandibular overhang lengths. Moreover, birds with intermediate length overhangs have the lowest feather mite loads. These results suggest that Darwin’s finches maintain an optimal beak morphology to effectively control their ectoparasites.     

Individual selection,culture and manipulation of higher plant cells

Summary Due to the heterogeneity in morphology, physiological and morphogenetical capabilities of higher plant cells in mass culture, the development of methods for individually culturing defined cells seemed to be useful and necessary. Individual cell culture represents a powerful tool for studies on the physiology of different cell types, the analysis of differentiation programs, the genetic manipulation of plant cells and cell-cell interactions. An improved microculture system based on a computer-controlled set-up for the efficient selection, transfer and individual culture of defined higher plant cells until regeneration of whole plants is described. Related experimental approaches for individually manipulating higher plant cells under controlled conditions, such as electrofusion of defined pairs of protoplasts and subprotoplasts, cell reconstruction and intranuclear microinjection of protoplasts and karyoplasts — mainly performed with cells of the crop plant Brassica napus L. — are presented.This article is dedicated to the memory of H.-G. Schweiger, initiator and mentor of the experimental single cell approach reviewed herein     

Nucleotide sequence analysis of 95 kb near the 3' end of the murine T-cell receptor alpha/delta chain locus: strategy and methodology.

The nucleotide sequence of a region at the 3' terminus of the murine T-cell receptor alpha/delta chain locus is presented. This region, which encodes the constant region genes for alpha and delta chain polypeptides and all 50 joining gene segments for the alpha chain polypeptide, spans 94,647 bp and includes more than 50 noncoding sequence elements important for T-cell receptor gene rearrangement and expression. DNA sequencing of this region included complete analysis of two cosmid clones and five additional restriction fragments using a random subcloning approach with various manual and automated sequencing strategies. The automated sequencing strategies hold considerable promise for future large-scale DNA sequencing efforts.     

Oxidative and reductive metabolism by cytochrome P450 2E1.

We are constantly exposed to many potentially toxic chemicals. Most require metabolic activation to species responsible for cell injury. Although cytochrome P450 2E1 is only one of many different forms of cytochrome P450 that catalyze these reactions, it has an important role in human health as a result of being readily induced by acute and chronic alcohol ingestion. The enzyme efficiently catalyzes the low Km metabolism of compounds commonly used as solvents in industry and at home as well as components found in cigarette smoke, many of which are established carcinogens and hepatotoxins. As a result, there is the potential for increased risk to low level exposure to such chemicals while cytochrome P450 2E1 is induced. Many substrates have been identified for cytochrome P450 2E1. Of the 52 substrates for the enzyme identified in this review, the demethylation of N,N-dimethylnitrosamine and the hydroxylation of p-nitrophenol and chlorzoxazone are the most effective for monitoring the level of this enzyme. In addition to oxidative reactions, cytochrome P450 2E1 is also an efficient catalyst of reductive reactions. CCl4-induced hepatotoxicity is one of the best-documented cases for the participation of cytochrome P450 2E1 in a toxicologically important reductive reaction. The reduction of oxygen to superoxide and peroxide are also important reductive reactions of the enzyme and could be important in lipid peroxidation. However, the role of this reaction in vivo remains controversial.     

Cladistical analysis of primitive G-band sequences for the karyotype of the ancestor of the Cricetidae complex of rodents

Homologous segments identified by G-banding sequences of chromosomes of Peromyscus boylii, Neotoma micropus, Oryzomys capito, (Family Cricetidae) Rattus norvegicus, Melomys burtoni, and Apodemus sylvaticus (Family Muridae) were used to hypothesize a chromosomal condition for the cricetid ancestor. A critical assumption in proposing the primitive G-banding sequences for a given chromosome is that if the outgroup and ingroup taxa have a specific sequence, then the ancestor of the ingroup taxa also had that same sequence. Using this methodology, (chromosome numbers refer to proposed homology to the standardized karyotype for Peromyscus), we propose that: (1) the primitive banding pattern of chromosome 1 was identical to that of Neotoma; (2) the primitive patterns of chromosomes 2, 3, 4, 6, 7, 8, 9, 10, 11, and 12 were primitive banding patterns of 5 and 13 were undetermined; (4) a major portion of the banding patterns of 14 and X were present in the ancestral karyotype. Only the largest 14 autosomes and X were examined because the smaller elements had insufficient G-band definition to ensure reasonable accuracy. The karyotype ancestral to that of Peromyscus, Neotoma, and Oryzomys may be as above and the banding patterns of 5, 13, and 14 were acrocentric and identical to those shown for Peromyscus, Neotoma, and Oryzomys (Fig. 1). In the primitive karyotype, heterochromatin (C-band material) was probably limited to the centromeric regions. If the primitive karyotype is as described above, then it is possible to determine the direction, type, and magnitude of chromosomal evolution evident in the various cricetid lineages. Based on the available data, radiation from the ancestral cytotype is characterized by a nonrandom distribution of types of chromosomal changes. Within many genera, more rearrangements occur in the 14 largest autosomal chromosomes of some congeneric species than distinguish the proposed primitive conditions for the genera Peromyscus, Neotoma, and Oryzomys. It would appear that the extensive morphological radiation from the primitive cricetid ancestor as indicated by the presence of over 100 surviving genera within the family, was not accompanied by extensive karyotypic changes. The magnitude of chromosomal variation that accompanies speciation in these genera appears to range from no detectable chromosomal evolution to a radical reorganization of the genome.     

Determination of ochratoxin A in liquorice products using HPLC based analytical methods. Part I: proficiency test of methods commonly used by the confectionary industry

A European proficiency test series was accomplished on behalf of the CAOBISCO (Association of the Chocolate, Biscuit and Confectionery Industries of the EU) expert group on ochratoxin A to assess the performance of laboratories in measuring ochratoxin A in samples of various liquorice products. In addition, the impact of the extraction type (mainly with or without the use of halogenated solvents) was to be evaluated. Four different test samples (two liquorice powders and two liquorice pastes) were tested for sufficient homogeneity and distributed to 15 laboratories in 8 countries in Europe. The results were analysed using standard proficiency testing statistical procedures and laboratories were awarded z-scores on the basis of their reported results. The overall evaluation of the results shows a distinct variation between the participating laboratories. Based on a target standard deviation (σ-value) taken from the Horwitz equation, of the 14 laboratories that reported results, satisfactory results (z-score: |Z| ≤ 2.0) were obtained by 60% and 27% of the laboratories, respectively, for the two liquorice powders and by 93% and 53% of the laboratories, respectively, for the two liquorice pastes. Approximately equal numbers of laboratories used extraction types with and without the use of halogenated solvents. ANOVA testing of the results indicates there was no evident trend using different extraction solvents.     

Two-step synergism between the progesterone receptor and the DNA-binding domain of nuclear factor 1 on MMTV minichromosomes.

In contrast to its behavior as naked DNA, the MMTV promoter assembled in minichromosomes can be activated synergistically by the progesterone receptor and NF1 in a process involving ATP-dependent chromatin remodeling. The DNA-binding domain of NF1 is required and sufficient for stable occupancy of all receptor-binding sites and for functional synergism. Activation of purified minichromosomes is observed in the absence of SWI/SNF and can be enhanced by recombinant ISWI. Receptor binding to minichromosomes recruits ISWI and NURF38, but not brahma. We propose a two-step synergism in which the receptor triggers a chromatin remodeling event that facilitates access of NF1, which in turn stabilizes an open nucleosomal conformation required for efficient binding of further receptor molecules and full transactivation.